Single peptide is 2 peaks on RP-HPLC. Why?
Background
I'm using C18 reverse phase HPLC (TFA and acetonitrile gradients) to
separate fragments of a digested peptide. The problem is that the starting
peptide (16 amino acids, sequence NH2-DHLSDNYTLDHDRAIH-COO) detected at
206nm runs as two equal height overlapping peaks, not separable. It is
synthetic, but sequence analysis and mass analysis show it to be pure, ie
there is only one main species present. Also, I've had three separate
preparations made at two different sites, and the effect is seen in all of
them!
Trypsin treatment cleaves AIH from the C-terminal resulting in a single peak
corresponding to DHLSDNYTLDHDR and a small double peak corresponding to
AIH indicating that the variation lies in this AIH region.
The only suggestions I've had are that his-16 may be breaking open, or that
it's a conformational effect caused by an inflexible ile-his bond.
If anyone can confirm, deny, or offer an alternative I'd be grateful.
Thanks in advance
Paul
psansom at hgmp.mrc.ac.uk
Paul A Sansom tel: 081-748-9966 x4208
Department of Biochemistry fax: 081-748-5090
The Kennedy Institute of Rheumatology
6 Bute Gardens
Hammersmith
London W6 7DW